Presence and Formation Mechanism of Foodborne Carbonaceous

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Presence and Formation Mechanism of Foodborne Carbonaceous Nanostructures from Roasted Pike Eel (Muraenesox cinereus) Jingran Bi, Yao Li, Haitao Wang, Yukun Song, Shuang Cong, Chenxu Yu, Beiwei Zhu, and Mingqian Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02303 • Publication Date (Web): 14 Jun 2017 Downloaded from http://pubs.acs.org on June 15, 2017

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Journal of Agricultural and Food Chemistry

1

Presence

and

Formation

Mechanism

of

Foodborne

2

Nanostructures from Roasted Pike Eel (Muraenesox cinereus)

Carbonaceous

3 4

Jingran Biabc, Yao Liac, Haotao Wangac, Yukun Songac, Shuang Congac, Chenxu

5

Yua,d, Bei-Wei Zhuabc* and Mingqian Tanac*

6 7

a

8

Engineering Research Center of Seafood, Dalian 116034, China, B-W Zhu,

9

[email protected]; M. Tan, [email protected]; Fax: +86-411-86318657;

School of Food Science and Technology, Dalian Polytechnic University, National

10

b

11

China

12

c

13

Dalian 116034, China

14

d

15

Ames, IA 50011, USA

School of Food & Biological Engineering, Jiangsu University, Zhenjiang 212013,

Engineering Research Center of Seafood of Ministry of Education of China,

Department of Agricultural and Biosystems Engineering, Iowa State University,

16

ACS Paragon Plus Environment


17

Abstract

18

Food-borne nanostructures have gained more and more attention in recent years.

19

In this paper, the presence and physicochemical properties of carbonaceous

20

nanostructures (CNSs) from roasted pike eel (Muraenesox cinereus) were reported. The

21

monodispersed CNSs are strongly photoluminescent under the illustration of UV light

22

with a fluorescent quantum yield of 80.16%, and display excitation-dependent emission

23

behavior. The formation of CNSs is believed to go through a process of morphology

24

evolution, including polymerization, pyrolysis, nucleation, growth, emergence and

25

blossom. The optical properties of the CNSs were shown to be affected by the roasting

26

temperature. Furthermore, cellular uptake of the CNSs was investigated, and it is

27

shown that the CNSs were clearly absorbed into live cells, and they mainly distributed

28

within the cell cytoplasm, not in the cell nucleus. This work is among the very first

29

reports on CNSs present in roasted fish, providing valuable insights into formation

30

mechanism of such nanostructures, and showcases the bio-distribution of these

31

food-originated CNSs in live cells.

32 33

Keywords: carbonaceous nanostructures, food-borne, formation mechanism,

34

photoluminescent, bio-distribution

35 36


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Introduction

38

The recent decade has seen the rapid development of innovative nanotechnology

39

in many fields. In food industry, nanotechnology also provides a variety of potential

40

benefits.1, 2 However, nanotechnology-derived foods are also new to consumers, and it

41

sometimes causes public alarm and anxiety.3 The direct and/or indirect impacts of

42

nanoparticles (NPs) on human health are still being understood. Some reports showed

43

that NPs may infiltrate many body compartments, and interact directly with

44

macromolecules in the body.4,

45

different mechanisms, i.e., the induction of reactive oxygen species (ROS),

46

genotoxicity, morphological modifications, NPs degradation, and triggering

47

immunological effects.6-9 Also, NPs present different hazards from those of the same

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material in bulk forms.10 Moreover, due to the insufficient health risk assessments of

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NPs, evidences related to the general safety of NP-containing foods are still

50

inconclusive. Besides, attention should be paid to food-borne nanostructures that arise

51

during natural food processing, to characterize their chemical composition,

52

characteristics and formation mechanisms. However, a literature survey revealed that

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little effort has been made in this research area.

5

NPs may induce toxicity via a combination of

54

Food processing transforms raw ingredients, by physical or chemical means, into

55

more ready-to-eat forms, which typically involves activities such as roasting, boiling,

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broiling, frying, grilling, steaming and electromagnetic treatment.11 In processes that

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involve intensive heating, macromolecules such as proteins and starches can be

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broken down, while small molecules can react to form aggregates. In a way it mimic

59

the preparation processes of carbon NPs, which are generally classified into two



60

categories – “top-down” and “bottom-up”.12 The former involves breaking down

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large carbon structures by methods like arc discharge, laser ablation and

62

electrochemical oxidation, while the latter involves the synthesis from small

63

molecular precursors through combustion/thermal treatments, or supported synthetic

64

and microwave synthetic routes. Due to the similarity between heat-based food

65

processing such as roasting and carbon NPs synthesis, it has been hypothesized that

66

carbonaceous nanostructures (CNSs) with multi-colorful fluorescence could be

67

produced during food processing. It was first reported by SK et al. the presence of

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amorphous carbon NPs in food caramels.3 The metabolic stress of these carbon NPs

69

was studied in human mesenchymal stem cells through CYP1A and p53 gene

70

expression.13 We also reported the discovery of CNSs in several commercial

71

beverages14 and instant coffee15. However, the study of food-borne CNSs is still in its

72

infancy, there are gaps in current knowledge of how these CNSs are created from

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foods, due to insufficient understanding of the complicated reaction pathways in foods

74

under thermal processing.

75

Fishes are important resources of food proteins for human consumption

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worldwide. Among them, pike eel (Muraenesox cinereus) with strong muscle is a

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species of eel widespread across Indo-Pacific, Southeast Asia, Indian Ocean and

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Australia. It is regarded as one of the most important functional food-fish species with

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high contents of proteins and polyunsaturated fatty acids. Roasting is a common

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cooking method for pike eel, in which hot air from an open flame, an oven, or other

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heat sources envelopes the food.16 It enhances flavor through caramelization and

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Maillard browning on the surface of the food. It is reasonable to believe that the


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intensive heating during roasting could also produce carbonaceous or self-assembled

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nanostructures with some unique physicochemical properties.

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In this study, the presence of food-borne CNSs in roasted pike eel (Muraenesox

86

cinereus) was confirmed for the first time, and the resulted CNSs were characterized.

87

The effects of heating temperature on the physicochemical properties of the CNSs

88

were investigated, and the formation mechanisms of these multi-colorful CNSs were

89

described. The uptake and bio-distribution of the CNSs in live cells were assessed.

90

These findings may help to draw attention to the CNSs in roasted foods and their food

91

safety implications.

92

Experimental

93

Preparation of CNSs from the roasting pike eel

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Livepike eel (Muraenesox cinereus) was purchased at a local fish market in

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Dalian, China on the day of the experiment. Upon arrival at the laboratory, pike eel

96

was immediately eviscerated and deboned. For preparation of CNSs, the raw fish

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fillets were cut into 1 ×1 × 0.5 cm pieces and roasted in a Self Cooking Center steam

98

oven (Rational, SCC-WE-101, Bavaria, Germany) at various temperature (160, 200,

99

230, 260, 300 °C) for 30 min. Then, the roasted samples were mixed with ethanol

100

(100%) (w/v=1/1) and vigorously stirred for 24 hours. After filtering, the sample was

101

evaporated to remove the ethanol as the solvent by vacuum distillation, and

102

re-dissolved in ultrapure water. Subsequently extracted with chloroform to remove the

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liposoluble constituent, the aqueous solution was transferred into a dialysis bag with

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molecular cutting-off of 3.5 kDa, and was dialyzed against ultrapure water for 4 days



105

to remove residues. The dialysis solution was collected and freeze-dried in a vacuum

106

oven. Finally, the CNSs powders thus obtained were saved for further

107

characterization. All of the reagents are analytical grade and were used without

108

further purification.

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Instrumentation and Characterizations

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Transmission electron microscopic (TEM) images were collected using a

111

transmission electron microscopy (JEM-2100, JEOL, Tokyo, Japan). Absorption

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spectra were recorded at room temperature on an UV−vis spectrophotometer (Lambda

113

35, Perkin Elmer, Cambridge, USA). Fluorescence spectra were measured by a

114

fluorescence spectrometer (F-2700, Hitachi, Tokyo, Japan). The Fourier transform

115

infrared spectroscopy (FTIR) spectra were analyzed in the KBr medium on a Frontier

116

FTIR spectrometer (Perkin Elmer, Norwalk, USA). X-ray diffraction (XRD) patterns

117

were recorded by a diffractometer (XRD-6100, Shimadzu, Kyoto, Japan) with CuK α

118

radiation (λ=1.54060 Å) from 5°~60° at 5° min-1 scanning speed. X-ray photoelectron

119

spectroscopy (XPS) spectra were used to characterize the chemical composition using

120

an X-ray photoelectron spectrometer (ESCALAB250, Thermo VG, Waltham, USA).

121

Bio-distribution

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Mouse osteoblasts cells (MC3T3-E1) was purchased from the Cell Bank of Type

123

Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells

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were maintained in a 10% serum containing DMEM supplemented with 10% fetal

125

bovine serum and 1% penicillin/streptomycin in a humidified atmosphere with 5%

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CO2 at 37 °C for 24 h. The cells were trypsinized and seeded in tissue culture plates at


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an initial cell density of 1×105 cells/well. Then the CNSs from the roasted pike eel

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(heated at 300 °C for 30 minutes) were introduced to the cells with the concentration

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of 1.5mg/mL. After incubated at 37 °C for 24 h, the cells were washed thoroughly

130

three times with PBS (500 µL each time) and kept in PBS for the optical imaging by

131

inverted Laser scanning confocal microscopy (SP8, Leica, Wetzlar, Germany) with

132

excitation wavelength of 405 nm, 488 nm and 543 nm for blue, green and red region

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images collection, respectively.

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Results and Discussion

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Characterization of the CNSs extracted from the roasted pike eel

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A schematic of the CNSs formation in pike eel roasted at 300 °C is shown in Fig.

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1A. The morphology of CNSs obtained after 30 min roasting was characterized by

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transmission electron microscopy (TEM) (as shown in Fig. 1B). Visual inspection

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suggests that the CNSs are spherical in shape and monodispersed with a narrow size

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distribution in the range of 1.75~4.25 nm and a maximum population at 2.75 nm (Fig.

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1C). In the high-resolution TEM (HRTEM) image (Fig. 1B insert and Fig. S1), most

142

particles are observed to be amorphous carbon-dot-like structures without any lattices.

143

The powder XRD spectrum shows a broad peak at around 2ߠ = 21.48° (002) which is

144

attributed to highly disordered carbon atoms (Fig. 1D). In the FTIR spectrum (Fig.

145

1E), a broad peak at 3390 cm−1 can be assigned to O-H stretching vibration of the

146

hydroxyl group and a harp peak at 2937 cm−1 reveals C-H bonding presented in the

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CNSs. The strong vibrational absorption peak of CNSs centered at 1663 cm−1 is

148

attributed to C=O stretching. Absorption bands at 1452 cm−1 is attributed to the C=C



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stretching mode of the polycyclic aromatic hydrocarbons. In addition, the peak at

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1048 cm−1 corresponds to the asymmetric stretching vibrations of C-O. These results

151

reveal that the molecular structures of the CNSs mainly contain hydrophilic and

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polycyclic groups, which are associated with the nanostructures or spontaneously

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polymerized products formed during the roasting process. Three peaks at 285, 399

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and 532 eV in the CD XPS spectrum correspond to carbon, nitrogen and oxygen,

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respectively (Fig. 1F). XPS elemental analysis revealed the composition of the CNSs

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to be C 68.28%, O 15.33%, and N 16.39%, thus indicating these CNSs are actually a

157

kind of N-containing CNSs. The nitrogen content of 16.39% suggests a protein origin.

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Fig. 1G shows the high-resolution C1s peak, which could be fitted into three peaks at

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284.9, 286 and 288.2 eV, that correspond to C=C, C-O-C, O-C=O bonds,

160

respectively. These data show that the CNSs are mainly composed of graphitic carbon

161

(sp2)

162

carbonyl/carboxylate groups at their surfaces.

and

carbon

defects

(sp3),

and

contained

abundant

hydroxy

and

163

(Fig. 1)

164

The CNSs are strongly photo luminescent with unique optical properties. Fig. 2

165

shows the UV-vis absorption and photoluminescence spectra. The UV-vis absorption

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peak at 278 nm and 335 nm are assigned to π-π* transition of C=C bond and ݊-ߨ∗

167

transition of the C=O groups present on the surface of CNSs. The major uniform size

168

of the sp2 clusters were in the CNSs even though these sp2 clusters doped in the sp3

169

matrix. The color of the CNSs in the aqueous solution was yellow, and a bright cyan

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color was observed under a portable UV lamp (λ=365nm) (inset of Fig. 2A). The

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maximum fluorescence emission (Emmax) of 465 nm was found under the excitation


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(Ex) wavelength of 405 nm. In addition, an excitation-dependent emission was

173

observed with red-shift towards the long excitation (Fig. 2B and Fig. 2C), showing

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multi-color nature of the CNSs. The emission intensity increased with increasing

175

excitation wavelength in range of 280~400 nm and then decreased gradually at 400 ~

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470 nm. This behavior suggests that the band gap of CNS is affected by its surface

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state, which is analogous to a molecular state; whereas the size effect is the result of

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quantum dimensions, both of which contribute to the complexity of the excited

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states.17 Accordingly, as an intrinsic characteristic parameter, the fluorescence

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lifetime (τ) indicates the period of excited state for CNSs prior to returning to its

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ground state. The decay of fluorescence emission was non-monoexponential for CNSs

182

(Figure 2D), and the lifetime measured by time-resolved fluorescence measurement

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was 7.17 ns, suggesting that it is the radiative recombination of the excitons that

184

induce the fluorescence.18 The calculated quantum yield was 80.16% with quinine

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sulfate as reference, much higher than most of synthetic CNSs, such as carbon dots

186

synthesized with glutathione and ascorbic acid (18.2%), citric acid as carbon source

187

after modification with L-tyrosine methylester (3.8%).19, 20

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(Fig. 2) Formation mechanism of CNSs during the roasting process

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Pike eel undergoing roasting at various stages (with different temperature for 30

191

min) were elaborately photographed (Fig. 3). The raw fish exhibited a touch of pink

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on the tightening muscle (Fig. 3A). When roasted at 160 °C for 30 min, the flesh

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turned into golden color (Fig. 3B). Large sized irregular microstructures were

194

observed under TEM (Fig. 3G and Fig. S2). They were only weakly fluorescent in



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aqueous solution (Fig. 3M). This may be due to the incomplete combustion of

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biopolymer at 160 °C, resulting in thermal-mediated polymerization, lipid oxidation

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or pyrolysis of organic matter. Carbohydrates, proteins and lipids in the fish would

198

undergo a spontaneous thermal reaction to form heterogeneous structures through

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thermally induced polymerization. In contrast, after exposure to higher temperature

200

(200 °C), and the fish flesh turned to brown and curled (Fig. 3C). Accordingly, the

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micro morphology was changed and a few nanoparticles (Fig. 3H and Fig. S3) with

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relatively weak fluorescence (Fig. 3N) were formed. We hypothesize that at this stage

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the adhering and clustering bio-polymers were gradually disintegrating through

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pyrolysis. CNSs started to emerge through seed-mediated nucleation. A series of

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complex spontaneous chemical reactions may occur at this stage to trigger the

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formation of carbonaceous or self-assembled nanostructures, similar to the previously

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reported formation of nano-dispersions of mesophases by self-assembly of

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carbohydrates, protein and lipid under heat treatment through the Maillard reaction.21

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Furthermore, when the fish was roasted at 230 °C, the surface of fish emerged slightly

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charred (Fig. 3D) and the bio-polymers continued to shrink as shown by TEM images

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in Fig. 3I and Fig. S4. The nano-sized clusters showed stronger fluorescence (Fig.

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3O). This may indicate that extreme pyrolysis at higher temperature could lead to the

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non-enzymatic browning and lipid oxidation, as reported in spontaneous

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carbonization processes.22 At even higher temperature of 260 °C, The flesh turned

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black and the charred area increased (Fig. 3E). More dot-like CNSs (Fig. 3J) were

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seen accompanied by some cluster substance, with bright blue fluorescence (Fig. 3P).

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This is probably due to the breakage of carbon-carbon bonds within fish flesh at


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higher temperature, resulting in more CNSs forming from the fragmented

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bio-polymer residues. When the temperature increased to 300 °C, the surface of fish

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was completely charred (Fig. 3F). Near mono-dispersed CNSs with particle size of

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1.75-4.25 nm were found in TEM images (Fig. 3K), which emit strong cyan

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fluorescence under the irradiation of the UV light (Fig. 3Q). The ethanol extracts of

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the raw fish and the one roasted below 230 °C in aqueous solutions were colorless,

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while those exceeded 230 °C were brownish. The color was notably deeper with the

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higher temperature (Fig. 3L, M, N, O, P, Q). No fluorescence was observed from the

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ethanol extracted solution of raw fish under the excitation of UV light (Fig. 3L), while

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the fluorescence became stronger as the processed temperature increased. Form these

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findings, the CNS formation during the pike eel roasting is concluded to include

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polymerization, pyrolysis, nucleation, growth and emergence and blossom stages.

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Further study is still needed to reveal the exact formation mechanism of the

231

fluorescent CNSs at molecular level.

232

(Fig. 3)

233

FTIR spectroscopy was used to further investigate the chemical transformation

234

of the CNSs during heating process. As shown in Fig. 4, the FTIR peak intensity of

235

the fluorescence substance from the roasted pike eel decreased than that of the raw

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fish. The broad adsorption peak at about 3418~3390 cm-1 is assigned to the O–H

237

stretching vibration, and the peak at 1663~1593 cm-1 is ascribed to C=O (carbonyl,

238

ester, or carboxyl) groups, which become stronger and wider with the increase of

239

temperature, suggesting that hydroxyl or carboxyl groups were formed in the reaction.

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In addition, the peak at 1466~1406 cm-1 corresponds to C=C groups vibration of



241

aromatic structure, and the peaks of 1155~1120 cm-1 and 1040~1048 cm-1 correspond

242

to C-O asymmetric stretching. The weakening of these two peaks with rising

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temperature indicates elevated pyrolysis reaction during the pike eel roasting. Thus,

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this result strongly implied that different temperatures resulted in a variety of

245

spontaneously formed nanostructures from protein, lipid and carbohydrates of fish

246

with different chemical groups on their surface.

247

(Fig. 4)

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The UV-vis absorption spectra (Fig. 5A and inset) shows only one absorbance

249

peak at approximately 235 nm for the fluorescence substance extracted from roasted

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pike eel at 160 °C, indicating the high carbonization level of the aromatic core for the

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restrained non-radiative recombination. As for the CNSs formed at 230°C, the peak at

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235 nm gradually fades away due to the enhanced carbonization level of the core and

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the decreased number of surface defects for stronger absorption. Increasing the

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temperature to 260°C, a shoulder at 335 nm emerges. Finally, a broad absorbance

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band at335nm appears when the temperature reached 300°C. Interestingly, both the

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maximum excitation (Exmax) and emission (Emmax) red-shifted as the roast

257

temperature increased (Fig. 5B), with the EXmax wavelength shifted from 350 nm to

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405 nm, and the Exmax wavelength shifted from 432nmto465nm (Table S1).

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Moreover, all of the samples (160~260 °C) showed an excitation-dependent emission,

260

as the emission peak red-shifts with declining fluorescence intensity with increasing

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excitation wavelengths (Fig. 5C, D, E and F). The quantum yields of samples

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collected from fishes roasted at 160, 200, 230, 260, 300°C were 12.86%, 31.35%,

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42.10%, 50.70% and 80.16%, respectively, with a continuously increased lifetime


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(Table S1). Clearly, both the quantum yields and the fluorescence lifetime were

265

strongly affected by the stability of excited-state, which was strongly correlated with

266

high reaction temperature.

267

(Fig. 5)

268

Another interesting observation was from the XPS characterization of the surface

269

composition (Fig. S6, S7, S8, S9). Three peaks at 285, 399 and 532 eV were observed

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among all samples from the pike eel roasted at different temperatures, which indicated

271

they contained carbon, nitrogen and oxygen, respectively. The relative abundance of

272

carbon (C1s) remained unchanged, while that of the oxygen (O1s) decreased, and the

273

nitrogen (N1s) increased, with rising roasting temperature (Table S2). The extreme

274

high roasting temperature accelerates the Maillard reaction, and may lead to the rise

275

of the relative nitrogen abundance.23 XPS also revealed the reduction in C=C content

276

at 284.9 ±0.2 eV from 47.76% to 35.41% as a result of pyrolysis reaction (Table S3).

277

However, the oxygen of O-C=O group at 288.2±0.3 eV increased from 17.86% to

278

27.75% with increased temperature.

279

The key to the observed excellent fluorescence behavior is believed to associate

280

with the effect of amines. The unpaired electrons of the amine groups may participate

281

electron-withdrawing and electron-accepting behaviors of functional groups, which

282

lead to the electron density to be increased and the band gap to be lowered.24 A

283

similar tendency exists as the size of the fluorescence substance increases at different

284

roast stages, which could result in a red-shift.21 Furthermore, high nitrogen abundance

285

in the CNSs produced at higher temperature, indicating possibly a higher amine

286

presence, may also be the reason for the higher quantum yields. It has been reported



287

that primary amines at the edges of fluorescence substance have higher occupied

288

molecular orbital than hydrogen-terminated groups, and the resonance between the

289

delocalized π orbital and the molecule orbital in amino groups may result in the

290

narrowing of the optical band gap.25

291

A model is proposed here to explain the fluorescence processes of the CNSs

292

(Fig. 6). For the CNSs from low temperature, weak fluorescence may be due to the

293

excited electrons mainly relaxed to the ground state through a non-radiative route,

294

with very few amino-based fluorophores vibration/rotation.26 At high temperature, the

295

high-nitrogen abundance in the CNS enhances the effect of nitrogen atoms on the

296

properties of the CNSs and strengthens the fluorescence characteristics. (Fig. 6)

297

298

Bio-distribution of CNSs in live cells

299

The uptake and bio-distribution of CNSs from roasted pike eel by live cells were

300

subsequently investigated. MTT assay was conducted to evaluate the cytotoxicity of

301

the CNSs. No cytotoxicity to mouse osteoblasts cells (MC3T3-E1) was recorded,

302

even with a high CNS concentration of 20.0 mg/mL, incubated at 37 °C for 24 h (Fig.

303

S14). Additionally, no morphological change between the CNS-treated cells and the

304

control ones was observed, as shown in Fig. 7. In vitro confocal microscopy showed

305

that the CNSs are mainly present within the cell cytoplasm, not in the cell nucleus,

306

similar to previously reported by our group.14, 15 Moreover, the CNSs within cells

307

could emit blue, green, and red fluorescence when excited at 405 nm, 488 nm, and

308

543 nm, respectively, due to the unique excitation-dependent characteristics. This

309

phenomenon was consistent with other reported work in fluorescent imaging of


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carbon dots, showing that the as-prepared CNSs are good multiple-color bioimaging

311

agents.27 However, in vivo assessment of risks and bio-distribution of these CNSs in

312

mammals is stilled needed.

313

(Fig. 7)

314

In summary, our current work demonstrated a food-borne CNSs specimen

315

generated during the roasting of pike eel (Muraenesox cinereus). The strongly

316

photoluminescent CNSs exhibit an excitation-dependent emission behavior. A

317

formation mechanism of the CNSs during the roasting is proposed, which includes

318

polymerization, dehydration, nucleation, aggregation, emergence and blossom. The

319

properties of the CNSs can be tuned by adjusting the roasting temperature. In

320

addition, the CNSs showed excellent biocompatibility and could easily enter into the

321

cytoplasmic region of MC3T3-E1 cells without any seriously imposing toxicity.

322

Acknowledgement

323

This work was supported by National Key Research and Development Project

324

(2016YFD0400404) and the National Nature Science Foundation of China

325

(31601389).

326

Appendix: Supplementary material

327

Conflict of interest

328

The authors declare that they have no conflict of interest.

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Figure captions:

417

Fig. 1 (A) Schematic illustration, (B) TEM image (inset: high resolution TEM

418

image, scale bar=10 nm), (C) corresponding particle size distribution, (D) XRD

419

pattern, (E) FTIR spectrum, (F) XPS spectrum and (G) high-resolution XPS spectra of

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C1s of the CNSs from the roasted pike eel heated at 300 °C.

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Fig. 2 Optical properties of the CNSs from the pike eel roasted at 300 oC. (A)

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UV-vis absorption spectra and fluorescence spectra of the CNSs. Insets show the

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photographs of the CNSs under daylight and a 365 nm UV lamp. (B) 3D color surface

424

map of the fluorescence spectra (300- 800 nm) at different excitation wavelengths

425

(310- 470 nm) of the CNSs. (C) Variation of the EM wavelength and intensity as a

426

function of EX wavelength. (D) Fluorescence decay curve of the CNSs.

427

Fig. 3 Schematic proposed formation mechanism of the CNSs from the roast

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pike eel. (A, B, C, D, E, F) photographs of the fish in various roast stages, (G, H, I, J,

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K) TEM images of the fluorescence substance extracted at different roast stages, (L,

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M, N, O, P, Q) photographs of the CNSs under daylight and a 365 nm UV lamp. The

431

arrows show the speculated formation processes of highly photoluminescent CNSs.

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Fig. 4 Evolutionary FTIR spectra of the fluorescence substance from roast pike eel at different temperatures by using the raw pike eel as a control.

434

Fig. 5 Optical properties of the fluorescence substance at different roast stages.

435

(A) UV absorption spectra of the pyrolysis products at different roast temperatures for

436

30 min. (B) Relationship between the Exmax and Emmax for the pyrolysis products at

437

different roast temperatures. 3D surface color maps of the emission spectra for the

438

pyrolysis products at (C) 160 oC, (D) 200 oC, (E) 230 oC and (F) 260 oC.


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Fig. 6 Schematic illustration for the proposed fluorescence processes of CNSs from the pike eel at low or high temperature.

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Fig. 7 Laser scanning confocal microscopy images of mouse osteoblasts cells

442

(MC3T3-E1) incubated with CNSs (300 oC) from the roast pike eel after incubation

443

for 24h.

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Fig. 1

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Fig. 7

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Table of Contents Graphic

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Presence and Formation Mechanism of Foodborne Carbonaceous

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